Whey Protein Inhibits Iron Overload-Induced Oxidative Stress in Rats

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1 J Nutr Sci Vitminol, 59, 98 5, 3 Whey Protein Inhiits Iron Overlod-Induced Oxidtive Stress in Rts Jungmi Kim, Hyun-Dong Pik, Yoh-Chng Yoon nd Eunju Prk, * Deprtment of Food nd Nutrition, Kyungnm University, Chngwon 63 7, Kore Food Science nd Biotechnology of Animl Resources, Konkuk University, Seoul 43 7, Kore (Received Septemer 7, ) Summry In this study, we evluted the effects of whey protein on oxidtive stress in rts tht were sujected to oxidtive stress induced y iron overlod. Thirty mle rts were ssigned to 3 groups: the control group (regulr [5 mg/kg diet] dose of iron% csein), iron overlod group (high [, mg/kg] dose of iron% csein, IO), nd whey protein group (high dose of iron% csein% whey protein, IOwhey). After 6 wk, the IO group showed reduction in the plsm totl rdicl trpping ntioxidnt prmeter nd the ctivity of erythrocyte superoxide dismutse nd n increse in lipid peroxidtion (determined from the proportion of conjugted dienes). However, whey protein meliorted the oxidtive chnges induced y iron overlod. The concentrtion of erythrocyte glutthione ws significntly higher in the IOwhey group thn in the IO group. In ddition, whey protein supplementtion fully inhiited iron overlod-induced DNA dmge in leukocytes nd colonocytes. A highly significnt positive correltion ws oserved etween plsm iron levels nd DNA dmge in leukocytes nd colonocytes. These results show the ntioxidtive nd ntigenotoxic effects of whey protein in n in vivo model of iron overlod-induced oxidtive stress. Key Words whey protein, iron overlod, oxidtive stress, ntioxidnt, DNA dmge Whey is y-product of csein precipittion, n importnt step in the mnufcture of cheese. It hs ecome vlule food ingredient ecuse of its excellent nutritionl vlue nd functionl properties. It constitutes pproximtely 85 9% of the volume of milk tht is used to mnufcture ripened cheese, nd it retins pproximtely 55% of the milk nutrients (, ). The mjor constituents of ovine whey protein (WP) re -lctogloulin (55 6%) nd -lctlumin (5 %). The minor constituents include ovine serum lumin, lctoferrin, immunogloulins, glycomcropeptides, phospholipoproteins, ioctive fctors, nd enzymes (3 5). Severl functionl nd iologicl ctivities of WPs hve een reported, such s opioid inhiition, inhiition of ngiotensin I-converting enzyme, ntihypertensive ctivity, immunomodultory ctivity, cteriosttic ctivity, nd downregultion of ftty cid synthesis in the liver (, 6 9). WP possesses ntioxidnt ctivity, which hs een recognized s the fctor responsile for the cheltion of trnsition metls y serum lumin nd lctoferrin, n iron-inding glycoprotein, s well s for the free rdicl scvenging ctivity shown y mino cids such s tyrosine nd cysteine ( 3). The in vivo ntioxidnt effects of WP hve een demonstrted in the hert tissue of iron-treted mice nd in the plsm nd liver tissue of vitmin E-deficient rts (4, 5). Mny pro-oxidnt drugs nd other chemicls hve * To whom correspondence should e ddressed. E-mil: pej@kyungnm.c.kr een implicted in the oxidtive stress nd cell injury tht result from the intrcellulr production of hrmful oxygen rdicls (6). Trnsition metls, such s iron nd copper, with their multiple oxidtion sttes re vitl for the cscdes of electron trnsfer rections. These metls prticipte in cellulr processes such s oxygen trnsport, photosynthesis, nitrogen fixtion, nd respirtion in most orgnisms (7 9). Iron is the most undnt trnsition metl in the humn ody; is primrily present in protein-ound forms, such s in heme nd non-heme proteins; nd plys crucil role in electron trnsfer nd oxygen utiliztion rections (). Iron exposure is directly ssocited with the pthogenesis of mny disorders, such s therosclerosis, cncer, nd inflmmtion, possily vi the production of rective oxygen species (ROS) (9). The results of recent in vitro nd in vivo (niml) studies hve shown tht certin dietry ntioxidnts protect ginst iron-induced oxidtive stress (, ). However, the ntioxidnt effect of WP on iron-induced oxidtive stress hs not een studied thoroughly. In this study, we investigted the effect of WPs on oxidtive stress in rts sujected to iron overlod-induced oxidtive stress. Mterils nd Methods Preprtion of WP. WP (4% protein) ws prepred from fresh mozzrell cheese whey in the lortory y ultrfiltrtion. Animl nd diets. Eight-week-old mle Sprgue Dwley rts (n53) were purchsed from Smtko Inc. (Osn, Kore). They were housed nd cred for in ccor- 98

2 Antioxidnt Effect of Whey Protein 99 Tle. Composition of the experimentl diet. (g/ g diet) Control IO IOWP Csein WP Cornstrch Sucrose Corn oil Cellulose Vitmin mixture Minerl mixture Choline itrtrte dl-methionine Butylted hydroxytoluene... Iron (FeSO 4 7H O) Totl Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. The AIN 76 vitmin mixture contined (in g/kg of mixture) the following: thimine HCl,.6; rioflvin,.6; pyridoxine HCl,.7; nicin, 3.; d-clcium pntothente,.6; folic cid,.; d-iotin,.; cynocolmin (vitmin B ),.; dry vitmin A plmitte (5, U/d),.8; dry vitmin E cette (5 U/d), ; vitmin D 3 triturtion (4, U/g),.5; mendione sodium isulfite complex,.5; finely powdered sucrose, The AIN 76 minerl mixture contined (in g/kg of mixture) the following: clcium phosphte, disic, 5; sodium chloride, 74; potssium citrte, monohydrte, ; potssium sulfte, 5; mgnesium oxide, 4; mngnous cronte (43 48% Mn), 3.5; ferric citrte (6 7% Fe), 6; zinc cronte (7% ZnO),.6; cupric cronte (53 55% Cu),.3; potssium iodte,.; sodium selenite,.; chromium potssium sulfte,.55; finely powdered sucrose, 8.3. dnce with the Guide for the Cre nd Use of Lortory Animls (3). All experimentl protocols for niml cre nd use were pproved y the Institutionl Animl Cre nd Use Committee t Kyungnm University, Chngwon. The rts were cclimtized to the niml fcility room in Kyungnm University for wk. They were housed individully ( C; : -h lightdrk cycle) with free ccess to commercilly prepred pelleted diet nd wter. The rts were then rndomly divided into 3 groups of nimls ech nd fed either stndrd diet (control group), stndrd diet supplemented with.% ferrous iron (iron overlod group, IO), or stndrd diet supplemented with.% ferrous iron% whey protein (IOwhey group) for 6 wk (Tle ). We fed.5 g Fe (FeSO 4 7H O)/kg dry mtter (DM) to the control group nd 9.9 g Fe/kg DM to the iron-overlod group, ccording to the method descried y Lfy et l. (). The nimls were monitored dily for generl helth, nd ody weights were recorded every week for the durtion of the study. At the end of the experimentl period, the rts were nesthetized with ethyl ether. The colon ws removed for the comet ssy, nd lood ws collected from the dominl rtery in heprinized sterile tue. Whole lood ws freshly prepred for the comet ssy. Plsm ws otined from the lood smples y centrifugtion (,5 rpm for 3 min) nd stored t 8 C until required for further nlysis. Erythrocytes were wshed 3 times with isosmotic phosphte-uffered sline (PBS, ph 7.4) nd resuspended to the originl volume. The erythrocyte suspensions were frozen t 8 C until they were required for the finl nlysis. The livers were removed from the rts nd wshed with ice-cold sline, t which time they were stored t 8 C efore nlysis. Totl plsm iron. The totl plsm iron concentrtion ws determined using n ssy kit (QuntiChrom TM Iron Assy Kit, BioAssy Systems, Hywrd, CA) sed on the ferrozine spectrophotometric technique. Plsm totl rdicl trpping ntioxidnt potentil. The plsm totl rdicl trpping ntioxidnt potentil (TRAP) ws mesured using modifiction of the photometric method developed y Rice-Evns nd Miller (4). The method used for mesuring ntioxidnt ctivity is predicted on the ntioxidnt-induced inhiition of the sornce of the rdicl ction of, -zinois (3-ethylenzothizoline 6-sulfonte) (ABTS ). The ABTS rdicl ction is formed y interction etween ABTS (5 mm) nd the ferryl myogloin rdicl species, which, in turn, is generted y the ctivtion of metmyogloin (.5 mm) y H O (75 mm). Ten microliters of smple/uffer/trolox-stndrd ws dded to tues contining 4 ml of PBS, ml of metmyogloin, nd 4 ml of ABTS nd mixed y vortexing. The rection ws initited y the ddition of 7 ml of H O. After 6 min of incution, the sornce ws mesured t 734 nm using spectrophotometer. Vlues hve een expressed in terms of Trolox equivlent ntioxidnt cpcity (TEAC) nd defined in terms of the millimolr concentrtion of the Trolox ntioxidnt cpcity of clirtion curve. Bseline levels of conjugted dienes in low-density lipoprotein. Bseline low-density lipoprotein (LDL)-conjugted diene levels were determined ccording to the methods outlined y Ahotup et l., with slight modifictions (5). Plsm ( ml) ws dded to 7 ml of heprin citrte uffer (.64 m trisodium citrte; 5, IU/L heprin; ph 5.5), nd the suspension ws incuted for min t room temperture. The insolule lipoproteins were then sedimented y centrifugtion t,5 rpm for min. The pellet ws resuspended in ml of. m N-phosphte uffer contining.9% NCl (ph 7.4). Lipids were extrcted from ml of the LDL suspension with chloroform-methnol ( : ), dried under nitrogen tmosphere, dissolved in cyclohexne, nd nlyzed spectrophotometriclly t 34 nm. Oxidtion during smple preprtion ws prevented y the ddition of ethylenediminetetrcetic cid (EDTA). Plsm lipid-solule vitmins. Plsm concentrtions of retinol, crotenoids, -tocopherol, nd coenzyme Q were determined simultneously y reversed-phse high pressure liquid chromtogrphy (RP-HPLC) ccording to the method reported y Jko nd Elmdf (6). Briefly, plsm proteins were precipitted with ethnol,

3 Kim J et l. nd lipids were extrcted with n-hexne. After evportion, the dry residue ws dissolved with 5 ml of methnol-dichloromethne (85 : 5, v/v) nd mixed. One hundred microliters of this solution ws injected into gurd-column (Merck LiChrospher RP8 [ mm], 534 mm). Smples were run t flow rte of. ml/min on Summit TM HPLC system (Dionex, Sunnyvle, CA). Asorption ws monitored t 35 nm for retinol, 95 nm for -tocopherol, 45 nm for crotenoids, nd 7 nm for coenzyme Q. Concentrtions were clculted from the res under the curve y using n externl clirtion curve. Erythrocyte ntioxidnt enzyme ctivities. Glutthione-peroxidse (GSH-Px) ctivity ws determined ccording to the method descried y Beutler (7). Ten microliters of erythrocytic hemolyste ws dded to ml of m Tris-HCl 5 mm EDTA uffer (ph 8.), ml of. m glutthione, ml of U/mL glutthione reductse, nd ml of mm NADPH. H O ws dded to otin finl volume of ml. After min of incution t 37 C, the rection ws initited y the ddition of ml of t-utyl hydroperoxide, nd the sornce ws mesured t 34 nm. The rection ws run for 9 s, nd the oxidtion of NADPH ws monitored y the chnge in the A 34 nm /min vlues. Ctlse (CAT) ctivity ws mesured ccording to the method developed y Aei (8). One hundred microliters of erythrocytic hemolyste ws dissolved in 5 ml of 5 mm phosphte uffer (ph 7.), nd ml of the mixture ws dded to cuvette. The rection ws initited y the ddition of ml of 3 nm H O t C. The H O decomposition rte ws mesured t 4 nm for 3 s y using spectrophotometer. The ctivity of superoxide dismutse (SOD) ws ssyed in the erythrocyte suspension y using the procedure reported y Mrklund nd Mrklund (9). Briefly, 3.5 ml of wter, ml of ethnol, nd.6 ml chloroform were dded to 5 ml of the hemolyste. After the mixture ws centrifuged t 3, rpm for min, vrious dilutions were prepred from the superntnt. After the diluted solutions were incuted t 37 C for min, ml of pyrogllol ( mm) ws dded to ech dilution. The rection ws monitored spectrophotometriclly t 3 nm for min. A unit of enzyme ws defined s the mount tht inhiited the utoxidtion of pyrogllol y 5%. The ctivity of glutthione-s-trnsferse (GST) ws ssyed y the method reported y Hig et l. (3), using. mm -chloro-,4-dinitroenzene (CDNB) s the sustrte. Fifty microliters of hemolyste ws dded to,935 ml of. m phosphte uffer contining. m NH PO 4,. m K HPO 4, nd 3 ml of. m glutthione (GSH). CDNB (5 ml) ws dded to strt the rection. The chnges in opticl density were recorded t 34 nm for 3 min. The enzyme ctivity ws clculted using n extinction coefficient of 9.6 mm cm. The hemogloin contents of the erythrocytic hemolystes were determined using n ssy kit (BCS, Anyng, Kore). The ctivities of SOD, CAT, GSH-Px, nd GST were clculted in terms of U/g H. The GSH concentrtions in erythrocyte suspensions were mesured using GSH ssy kit (Cliochem, Sn Diego, CA). DNA dmge determintion y the lkline comet ssy. Colon cells were isolted from dissected colon tissue for cytotoxicity nd genotoxicity nlyses. The lkline comet ssy ws conducted ccording to the protocols estlished y Singh et l. (3), with little modifiction. Frosted slides (Fisher Scientific) were prepred with sl lyer of.5% norml melting grose s follows: 5 ml of whole lood or colon cell suspension (3 9 colon cells/l) ws mixed with 75 ml of.7% low melting grose (LMA) nd then dded to the slides. The slides were covered with cover slips nd kept in refrigertor for min. The cover slips were then removed, nd top lyer of 75 ml of.7% LMA ws dded efore plcing the slides (with cover slips) in refrigertor gin for min. After removl of the cover slips, the slides were immersed in jr tht contined cold lysing solution (ph.) consisting of.5 m NCl, mm EDTA, mm Tris, nd % sodium lurylsrcosine. Susequently, % Triton X- nd % DMSO were freshly dded to the solution, which ws then stored in refrigertor for h. After lysis, the slides were plced in horizontl electrophoresis tnk (Threeshine Co. Ltd., Dejeon, Kore). The slides were covered with fresh lkline uffer (3 mm NOH, mm N EDTA, ph 3.) nd mintined t 4 C for 4 min. DNA ws electrophoresed y pplying n electric current of 5 V/363 ma for min t 4 C. The slides were wshed 3 times with neutrlizing uffer (.4 m Tris, ph 7.5) for 5 min t 4 C nd then treted with ethnol for nother 5 min. All the steps following the lysis tretment were undertken in drkness in order to prevent dditionl DNA dmge. Fifty microliters of ethidium romide ( mg/ml) ws dded to ech slide, nd the slides were exmined using fluorescence microscope (LEICA DMLB, Wetzler, Germny). Imges of rndomly selected cells (5 cells from ech of replicte slides) were nlyzed for ech suject. Mesurements were mde with imge nlysis softwre (Komet 5., Kinetic Imging, Liverpool, UK) to determine the percentge of DNA in the til (til intensity). Sttisticl nlysis. Dt were nlyzed using the SPSS pckge for Windows (Version, SPSS Inc., Chicgo, IL). Vlues re expressed in terms of men6stndrd error (SE). The dt were evluted y one-wy ANOVA, nd the difference etween the mens ws ssessed using Tukey s test when the F vlue ws significnt. The differences were considered significnt t p,.5. Person s correltion ws used to evlute the ssocitions etween prmeters. Results Food intke, weight gin, nd orgn weight During the experiment, no signs of tretment-ssocited dverse effects were oserved in the clinicl ppernce of the nimls. No differences in weight gin, food intke, or food efficcy rtio were oserved mong the 3 groups (Tle ). The significnt increse in reltive

4 Antioxidnt Effect of Whey Protein Tle. Effects of whey protein on ody weight gin, food intke, nd FER in rts fed n iron overlod diet. Control IO IOWP Weight gin (g/d) ns, Food intke (g/d) ns FER ns Orgn weight (g/ g BW) Liver ns Hert.546., Kidney.966. ns Spleen.36. ns Vlues re the men6se for nimls in ech group. Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. FER, food efficiency rtio. 3 ns, not significnt. 4 The vlues in the sme row tht do not shre common superscript re significntly different t the p,.5 level. Plsm Fe (μg/dl) Fig.. Effects of whey protein on the plsm Fe concentrtion in rts fed n iron overlod diet. Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. Ech r represents the men6se vlue for nimls in ech group. Brs with different superscript letters re significntly different t p,.5 y Tukey s test. hert weight due to iron overlod ws corrected y whey protein supplementtion. Plsm totl iron The plsm iron concentrtion in the IO group ws significntly higher thn tht in the control group ( mg/dl vs mg/dl; p5.38) (Fig. ). Supplementing the diet with % whey protein lowered the plsm iron concentrtion y 7% in the IOWP group ( mg/dl; p5.8). Plsm ntioxidnt potentil nd lipid peroxidtion Iron overlod reduced the plsm TRAP, n indictor of totl ntioxidnt defense, nd incresed the proportion of plsm-conjugted diene, mrker of lipid peroxidtion (Fig. ). Whey protein supplementtion led to 3.7% increse in the TRAP vlues (p,.) nd significnt 4.7% reduction in the proportion of plsmconjugted diene (p,.5) in the IOWP group, when compred with the corresponding vlues in the IO group. Other ntioxidnt-relted prmeters in the plsm nd erythrocytes Tle 3 shows the effect of whey protein supplementtion on the plsm concentrtions of ntioxidnt vitmins nd the ctivities of erythrocyte ntioxidnt Plsm TRAP (mm) Plsm CD (μm) A B enzymes in rts fed n iron-rich diet. The erythrocyte SOD ctivity in the IO group ws significntly lower (44.8% lower) thn the ctivity in the control group. However, whey protein supplementtion significntly incresed the erythrocyte SOD ctivity. The IOwhey group showed significnt increse in the concentrtion of erythrocyte GSH compred with tht in the IO group. No significnt chnges were oserved in the plsm concentrtions of ntioxidnt vitmins or the ctivities of other erythrocyte enzymes. Fig.. Effects of whey protein on plsm totl ntioxidnt potentil (TRAP) nd lipid peroxidtion (conjugted dienes, CD) in rts fed n iron overlod diet. Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. Ech r represents the men6se for nimls in ech group. Brs with different superscript letters re significntly different t p,.5 y Tukey s test.

5 Kim J et l. Tle 3. Effects of whey protein on plsm ntioxidnt vitmin concentrtions nd erythrocytic ntioxidnt enzyme ctivities in rts fed n iron overlod diet. Control IO IOWP Plsm Retinol/TG* ns, g-tocopherol/tg*.56. ns Tocopherol/TG* ns Erythrocyte Ctlse (K/gH) ns GSH-Px (IU/gH) ns SOD (U/gH), c,3, , GST (U/gH) ns GSH (mm) Vlues re the men6se for nimls in ech group. Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. ns, not significnt. 3 Vlues in the sme row tht do not shre common superscript re significntly different t the p,.5 level. * Corrected y plsm triglycerides. A Til intensity in Leukocytes (%) B Til intensity in Colonocytes (%) Leukocyte nd colonocyte DNA dmge Figure 3 shows the effects of whey protein supplementtion on iron overlod-induced DNA dmge in the peripherl lood cells nd colon cells of rts. Iron overlod induced significnt increse in the proportion of strnd reks in leukocyte DNA (IO vs. control: vs %; p,.) nd colonocyte DNA (IO vs. c Fig. 3. Effects of whey protein on DNA dmge in leukocytes nd colonocytes in rts fed n iron overlod diet. Control: norml control group, IO: iron overlod group, IOWP: iron overlodwhey protein. Ech r represents the men6se vlue for nimls in ech group. Brs with different superscript letters re significntly different t p,.5 y Tukey s test. Til intensity in Leukocytes (%) Til intensity in Colonocytes (%) A B Plsm Fe (μg/dl) control:.6.9 vs %; p,.). In rts fed diet supplemented with % whey protein for 5 wk, the strong genotoxic effect of iron overlod ws reduced y 6% in leukocytes nd y 87% in colonocytes. Plsm iron concentrtions showed highly significnt positive correltion with DNA dmge in leukocytes (r5.456, p5.3) nd in colonocytes (r5.38, p5.4) (Fig. 4). Discussion r=.456 p=.3 r=.38 p= Plsm Fe (μg/dl) Fig. 4. Reltionship etween leukocytic DNA dmge (A), colonocytic DNA dmge (B), nd plsm Fe concentrtion. r5person s correltion coefficient. The im of this study ws to determine whether incresed oxidtive stress due to iron overlod could

6 Antioxidnt Effect of Whey Protein 3 e meliorted y dietry supplementtion with whey protein. We found tht dietry supplementtion with whey protein for 6 wk ws effective in meliorting the oxidtive stress induced y dietry iron overlod. Whey protein supplementtion significntly incresed the plsm TRAP vlues nd erythrocyte SOD ctivity nd decresed the levels of plsm lipid peroxidtion. Free iron ville from ferrous sulfte supplementtion minly exists s insolule ferric Fe(III) t physiologicl ph nd in the presence of oxygen (3). Since iron trnsport systems re specific for ferrous Fe(II), ferric iron is reduced y duodenl ferric reductse (33), thus llowing cells to tke up iron. Highly unstle ferrous iron (Fe ) ctlyzes the conversion of hydrogen peroxide to ROS, such s hydroxyl ( OH) nd superoxide (O ) rdicls, through Fenton-type or iron-ctlyzed Her- Weiss iochemicl rections. These ROS cn dmge importnt iomolecules, nmely lipids, proteins, nd DNA (34 36). The results of in vitro nd in vivo studies hve shown tht iron overlod cn enhnce oxidtive stress nd increse DNA strnd rekge nd the oxidtion of DNA ses (37, 38). Our results showed tht consumption of diet supplemented with.% ferrous iron for 5 wk cn significntly increse plsm iron concentrtions nd consequently oxidtive stress, which ws evident from the decrese in the plsm TRAP vlues nd erythrocyte SOD ctivity nd the increse in the levels of plsm lipid peroxidtion. The oxidtive stress induced y iron overlod cused DNA dmge in peripherl leukocytes nd colonocytes; the dmge oserved ws lmost 5 times tht in the control group. Moreover, significntly positive correltion etween the plsm iron concentrtion nd the level of DNA dmge ws oserved in leukocytes nd colonocytes. Our findings re consistent with the findings of Rehmn et l. (39), who reported tht dietry supplementtion with iron sulfte nd vitmin C incresed the level of oxidtive dmge in the white lood cells of helthy humns. Furthermore, DNA dmge in the lymphocytes of ptients with thlssemi, genetic hemtologicl disorder chrcterized y n increse in iron concentrtion tht results from the impirment of hemogloin synthesis, ws 4 times higher thn tht oserved in the lymphocytes of norml individuls (4). The presence of unsored free iron moieties, which re produced s result of dietry supplementtion, hs een reported to increse the rte of free rdicl formtion in the colon of helthy volunteers to level tht cn cuse mucosl cell dmge (3). SOD ctivity in erythrocytes, whose potent enzymtic nd nonenzymtic ntioxidnt ctivities modify highly ROS into sustntilly less rective intermedites (4), ws decresed y iron overlod. Whey protein inhiited iron overlod-induced dmge to DNA in leukocytes nd colonocytes. Brtfy et l. (4) reported n in vivo ntioxidnt effect of whey protein; they showed tht mice receiving iron tretment ( mg/dl of iron dextrn, i.p.) supplemented with whey protein hd significntly lower levels of cytotoxic ldehydes nd higher levels of glutthione peroxidse (GSH-Px) nd GSH in hert tissue thn mice treted with iron lone. Zommr et l. (5) showed tht whey protein supplementtion increses the concentrtions of -tocopherol nd GSH in the plsm nd liver, respectively, wheres it decreses lipid peroxidtion in the liver of rts fed diet low in vitmin E. The mechnism underlying the ntioxidnt effects of whey protein on iron overlod-induced oxidtive stress my involve the iron-chelting properties of whey protein nd led to incresed iron excretion in urine nd feces. This ssumption is supported y the significnt reduction in plsm iron concentrtions in the IOWP group oserved in the present study. Lctoferrin, mjor component of whey protein, is elieved to chelte trce metls, including iron (6, 4). As reported y Bihel nd Birlouez-Argon (43), the inding of iron to lctoferrin my decrese the rte t which hydrogen peroxide is converted to hydroxyl rdicl vi the Fenton rection, therey inhiiting the oxidtion of scoric cid nd tryptophn. Another possile mechnism for the ntioxidnt effect of whey protein involves its ility to scvenge free rdicls. The results of in vitro studies showed tht -lctogloulin nd serum lumin, which re predominnt proteins in whey, contin free sulfhydryl group tht scvenges free rdicls, therey inhiiting lipid peroxidtion (6, 44). The cysteine-rich proteins in whey, which id in the synthesis of GSH, potent intrcellulr ntioxidnt (45), could lso contriute to the ntioxidnt effect of whey. The results of some studies hve shown tht iron overlod induces GSH depletion in the serum, liver, nd hert tissue (9, 4). Although diet supplemented with.% ferrous iron did not pper to ffect the GSH levels in erythrocytes, supplementtion of n ironrich diet with whey protein enhnced GSH synthesis in erythrocytes y 3%. We elieve tht the incresed GSH concentrtion helped enhnce the cellulr defense mechnisms ginst ROS formtion tht is induced y iron overlod in the whey protein-supplemented group. In conclusion, the findings of our study of n in vivo model of oxidtive stress induced y iron overlod clerly indicte tht whey protein protects ginst ironmedited ROS formtion nd therey protects ginst DNA dmge in white lood cells nd in the colon. Further nlyses of the ntioxidnt sttus in other orgns (e.g., liver nd hert) re required to understnd the exct mechnism underlying the protective effects of whey protein. Acknowledgments This work ws supported y grnt from the Koren Rurl Development Administrtion (ARPC). REFERENCES ) Rutherfurd-Mrkwick KJ, Gill HS. 5. 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